Our group has been working for some time now – since 2006, in fact – on investigating the feasibility of providing South (and southern) Africa with emergency response pandemic influenza vaccines. The research was initiated after the Virology Africa 2005 conference that Anna-Lise Williamson and I organised in the Cape Town Waterfront in November of that year – when a senior WHO official warned us in his talk that “…if a pandemic hits, you are on your own: no-one will give you any vaccine”.

A group of us sat down afterwards, and discussed the feasibility of looking at emergency response vaccine(s), given that we had no capability in the whole of Africa to make flu vaccines. Anna-Lise and I put together a proposal, with the highly pathogenic avian H5N1 influenza A as a target, which was funded on a once-off one-year basis by the Poliomyelitis Research Foundation (PRF) here in SA for 2006 – and then again by the PRF as a three-year Major Impact Project (MIP) from 2008-2010, and subsequently to a lower level by both the PRF and the Medical Research Council of SA. What made it all the more impressive for a South African project was that we had proposed expressing a protein-based vaccine in plants – quite a revolutionary prospect at the time, but something that followed on from the highly successful production of Human papillomavirus virus-like particles by transient expression in Nicotiana benthamiana by James Maclean, working as a postdoc in our lab at the time.

However, some of the most important work was done early: James was very quick to get the haemagglutinin (HA) gene for the A/Vietnam/1194/2004 strain of H5N1 synthesised by GeneArt in Germany, and cloned into the same Agrobacterium tumefaciens plant expression vectors from Professor Rainer Fischer’s lab in Aachen, Germany, that had been used for HPV. His initial work showed that large amounts of HA protein could be produced, both as soluble protein which lacked a membrane localisation domain, and as the membrane-bound form. This work formed the basis for a patent application on the transient expression of H5 HA that has now been granted.

Subsequently, when the PRF MIP started, we employed Dr Elizabeth (Liezl) Mortimer and Ms Sandiswa Mbewana to further the work: with collaborators from the National Institute for Communicable Diseases (NICD) in Johannesburg and State Veterinary Services in Stellenbosch, this investigated transient and transgenic expression of soluble and membrane-bound forms and their immunogenicity, as well as a DNA vaccine consisting of the HA genes cloned into Tomas Hanke’s pTH vector.

What we had managed to show was that we could get excellent production of the H5 HA in both soluble and bound forms, and that especially the membrane-associated form of the protein was highly immunogenic, and elicited antibodies in experimental animals that were appropriately neutralising, indicating its suitability as a vaccine candidate.

Now this all happened despite our running out of money AND Liezl leaving to have a baby…and then we managed to get another paper out of the work, this time on the DNA vaccine side of things.

We pitched this at the South African Journal of Science as a vindication of the faith in us by exclusively South African funding agencies – and managed to get the cover of the issue in which it appears, thanks to the truly excellent artwork of Russell Kightley from Canberra, Australia. Front AND back covers, as it happens…!

And this all made Sandiswa Mbewana, who is now a PhD student on another project, very happy:

The new international conference on virus-like particles and nano-particles (VLPNPV) took place in Cannes, France at The Novotel Montfleury Hotel from the 28th to the 30th of November 2012. The scope of the conference included virus-like particles (VLPs), the plant-based expression of VLP vaccines as well as expression and optimisation of VLPs.

Other topics included in the conference were:

VLP platform delivery systems

VLP vaccines

Nano-particles and nano-particulate vaccines

A multitude of topics were covered during the conference and many of the talks pertained to the immunogenicity of the VLPs and nano-particles and how they compared with the immunogenicity of DNA or subunit vaccines.

Talks were given by researchers from companies such as Medicago, Mucosis, Pevion Vaccines and Novavax. These talks gave a perspective on factors that need to be considered when commercialising VLP/nano-particle vaccines and to be GMP compliant.

This talk was about a plant-made VLP against both pandemic and seasonal influenza- these vaccines are now in the clinical trial phase. What was especially interesting was the view from an industry point of view where expression had to be scaled up to produce large amounts of vaccine. The Medicago platform can synthesize and clone approximately 100 gene constructs in two weeks, they can prepare 100 bacterial cultures per week and they have automated infiltration where 200 plant transformations can be performed per day and 150 VLP engineering approaches can be tested in one week. For influenza Medicago tested 48 different infiltration approaches in one day for HA, NA, M1, M2 as well as P1 Gag and HGalT. Medicago has been able to produce 10 million doses of HA VLPs in just one month.

This group made empty Cowpea Mosaic Virus (CPMV) VLPs that contained no RNA. CPMV VLPs are versatile nanoparticles to which organic, inorganic and biological molecules can be bound. The empty nature of the particle means that they can be used as carrier molecules for therapies; this could prove to be potentially useful as a cancer-treatment therapy. The system is advantageous because of the lack of RNA which makes the particles non-infectious and no bio-containment is needed for the production of these VLPs.

Immunogenicity of VLPs: an immunological perspective

Martin Bachmann (University of Zurich, Zurich, Switzerland)

Background was given from immunological point of view about what makes VLPs so immunogenic. Three properties contribute to the immunological properties of VLPs (1) their size, (2) the repetitiveness of the particle capsid which provides multiple sites for antibody binding and (3) TLR ligands – the particle can be disassembled, the RNA removed and replaced with a TLR ligand to enhance immunogenicity. Also, the size of VLPs is optimal for drainage to the lymph nodes.

Immunogenicity optimization strategies for public-sector development of vaccines: the critical role of optimizing the antigen.

Martin Howell Friede (WHO, Geneva, Switzerland)

This talk was about looking at VLPs from the vaccine development view. Monomeric antigens are not very immunogenic; therefore adjuvants were developed and came into use. For an efficient vaccine the antigen must be multimeric as antigen alone is insufficient to be immunogenic without adjuvant. Two factors have to be considered when producing a vaccine for FDA approval; (1) optimise the antigen before using an adjuvant, (2) use an adjuvant that has already been approved by the FDA. VLPs as vaccines provide the potential for immune-stimulation without the addition of adjuvant as the multimeric presentation of the antigen will enhance its immunogenicity.

Enhancing the immunogenicity of VLP vaccines

Richard W. Compans (Emory University, Atlanta, Georgia, USA)

This talk highlighted strategies which could be used to enhance the immunogenicity of VLPs.

Increase the breadth of immunity by enhancing responses to conserved antigens/epitopes

Increase the amount of antigen incorporated into VLPs

Incorporate the adjuvant into the VLPs as part of the structure

See also:

Ye et al (2011) PLoS One 6(5): e14813

Wang et al (2008) J Virol

Innate and adaptive responses to plant-made VLP vaccines

Brian Ward (McGill University, Montreal, Quebec, Canada)

Brain Ward is also the medical officer at Medicago. Humans rarely react to plant proteins/antigens. The plant glycans fucose/xylose at the N-terminal is an allergen and can cause anaphylaxis in humans. During trial experiments with influenza no individuals developed IgE responses to plant glycans, therefore plant produced vaccine is safe. The H1 VLP induced long lasting memory multifunctional T-cell responses in humans.

Impressions of the conference:

The conference was well organised with leaders in the field presenting their work. Interaction with the delegates aid in building crucial networking opportunities and work relationships. The international arena is packed with new technology development allowing us the opportunity to learn and improve our own understanding of various concepts.

This conference proved to be an invaluable learning experience and I thank the NRF for this opportunity and for providing me with the funding to attend this conference. The exposure to conferences, especially those in the international arena, aid in the development of students and contribute to the quality of research that is conducted at UCT.

I am TRYING to write an eBook on influenza, which stubbornly refuses to be finished – as part of a sabbatical project, which finished in December 2010. So, like my History of Virology, I am trialling the material on you, the Web community. Enjoy / comment / be enlightened / whatever!

NEW NOTE: The ebook is finished, and is available here on the iBooks Store: please try it out (you can read it on any Mac / iPad / iPhone using the free iBooks app)

History of Influenza

While they were not recognised as such at the time, major or pandemic outbreaks of influenza disease have occurred throughout recorded history. Medical historians have used contemporary reports to identify probable influenza epidemics and pandemics from as early as 412 BCE – and the term “influenza” was first used in 1357 CE, describing the supposed “influence” of the stars on the disease. The first convincing report of an epidemic of the disease was from 1694, and reports of epidemics and pandemics in the 18th century increased in quality and quantity.

The first pandemic that historians agree on was in 1580: this started in Asia, and spread to Africa, took in the whole of Europe in 6 months, and even got to the Americas. Subsequent pandemics with significant death rates occurred in 1729 and 1781-2; there was a major pandemic in 1880-1883 that attacked up to 25% of affected populations, and another in 1898-1900 that was probably H2N2. There is an excellent account here of the first “modern” pandemic, in 1890 – or at least, the first one to be followed essentially in real time via newspapers.

Influenza A pandemics in modern times. * = probably reintroduced from a laboratory from the H1N1 circulating from 1918 until 1957.

The “Spanish Flu” Pandemic 1918-1920

While the first reports of this pandemic were from Spain, this was largely because theirs was possibly the only uncensored press in Europe at the time because of the 1914-1918 War. In fact, it seems generally accepted that the virus originated in the United States, possibly in a military camp, and was then taken via infected personnel travelling by troop transport, to France by April 1918. The virus spread quickly across Europe, and via troop transports again to northern Russia, north Africa and India. Further spread then occurred, to China, New Zealand and ThePhilippines, all by June 1918.

Initially, there was nothing unusual: infections spread quickly for a while and then declined, and death rates were not higher than in previous pandemics. However, from August 1918 – marked by a ship-borne outbreak in Sierra Leone in west Africa – the virus seemed to have become markedly more virulent, and the death rate is supposed to have increased 10-fold. The virus quickly spread through Europe, to the USA, to India by October 1918, and to Australia by January 1919, all the while spreading through and around Africa.

Some countries had second and even third waves of infection, in 1918-1919 and 1919-1920. The pandemic was initially calculated as having killed some 20 million people: however, later estimates which took into account in particular the African, Indian and Chinese death tolls have increased the death toll to at least 50 million, and possibly up to 100 million.

The virus probably infected over one third of the humans alive at the time, with a casemortality rate of up to 5%. Some regions, like Alaska and parts of Oceania, had death rates of up to 25% of the total population. By contrast, the normal mortality rate for seasonal flu is 0.1 – 0.3% of those infected.

The pandemic was unusual in that it seemed to affect mainly young adults: The graph showscase mortality rates in percent for pneumonia and influenza combinedfor 1918-1919, and for seasonal influenza for 1928-1929, for different age groups. The “W” shape for the 1918-1919 figures is most unusual; the later seasonal data show a far more usual “U” curve. The green line shows whatcould have happenedif – as is suspected – people over 30 had not had some immunity to the virus, due to prior exposure to the H1 and/or N1 – possibly during the 1880 or 1893 pandemics.

Although secondary bacterial infections of the lungs were common in fatal cases in 1918, and contributed significantly to mortality, there were also many cases of rapid death where bacterial infection could not be demonstrated – so these were due to a so-called “abacterial pneumonia”. Incidentally, the archiving of pathology specimens from especially military cases in the USA proved invaluable in “viral archeology” studies as late as 1997.

Discovery of Influenza Virus

As early as 1901, investigators had shown that the agent of fowl plague was a “filterable virus”: however, this was not linked to human disease, as it was only shown to be an influenza virus in 1955.

Charles Nicolle and Charles Lebailly in France proposed in 1918 that the causative agent of the Spanish Flu was a virus, based on properties of infectious extracts from diseased patients. Specifically, they found that the infectious agent was filterable, not present in the blood of an infected monkey, and caused disease in human volunteers. However, many scientists still doubted that influenza was a viral disease.

A paper presented in 1918 to the Academie Francaise, describing the influenza agent as a filterable virus

In 1931, Robert Shope in the USA managed to recreate swine influenza by intranasal administration of filtered secretions from infected pigs. Moreover, he showed that the classic severe disease required co-inoculation with a bacterium – Haemophilus influenza suis – originally thought to be the only agent. He also pointed out the similarities between the swine disease and the Spanish Flu, where most patients died of secondary infections.

Pigs in the USA and elsewhere probably caught the H1N1 “Spanish Flu” from people – and it has circulated in them continuously until the present day

Patrick Laidlaw and others, working in the UK at the National Institute for Medical Research (NIMR), reported in 1933 that they had isolated a virus from humans infected with influenza from an epidemic then raging. They had done this by infecting ferrets with filtered extracts from infected humans – after an observation that ferrets could catch canine distemper – and then found that ferrets could transmit influenza to investigators by sneezing on them! The “ferret model” was very valuable, as strains and serotypes of influenza virus could be clinically distinguished from one another. Their serotype was named “influenza A”, and it was later typed as H1N1: this virus was a direct descendant of the Spanish flu virus, and had circulated in humans since 1918. It was the same subtype, incidentally, as that isolated by Shope from pigs.

Frank Macfarlane Burnet from Australia in 1936 showed that it was possible to do “pock assays” for influenza virus on the chorioallantoic membranes of fertilised chicken eggs, and subsequently said that:

“It can probably be claimed that, excluding the bacteriophages, egg passage influenza virus can be titrated with greater accuracy than any other virus.”

This finding led directly to the development of the first influenza A vaccine – a killed virus preparation made in eggs – by Thomas Francis in the USAin late 1943. He had earlier, in 1940, isolated the first influenza B, which was made into a vaccine by 1945. It was then clear that seasonal influenza was caused by two viruses: the A H1N1 type, and influenza B.

The “Asian Flu” of 1957-1958

After the influenza pandemic of 1918-1920, influenza went back to its usual seasonal pattern – until the pandemic of 1957. This started with the news that an epidemic in Hong Kong had involved 250 000 people in a short period. This was a unique event in the history of influenza, as for the first time the rapid global spread of the virus could be studied by laboratory investigation. The virus was quickly identified as an H2N2 subtype.

Except for people over 70, who had possibly been exposed to an influenza pandemic in 1898 – also probably a H2N2 pandemic – the human population was again confronted by a virus that was new to it – and again, the virus alone could cause lethal pneumonia. However, better medical investigation showed that chronic heart or lung disease was found in most of these patients, and women in the third trimester of pregnancy were also vulnerable.

The 1957 pandemic was the first opportunity for medical people to observe the vaccination response in the many people who had not previously been exposed to the novel virus. This was very different to the 1918 virus that had been circulating ever since, meaning that most people had no immunity to it at all. More vaccine was initially needed to give protective immunity than with the earlier type A vaccines. However, by 1960 as the virus recurred as a seasonal infection, immunity levels in the general population increased and vaccine responses were better, due to “priming” of the response by natural infection or first immunisation.

The death toll for this pandemic was around two million people – even though a vaccine was available by late 1957. Infections were most common among school children, young adults, and pregnant women in the early pandemic. Elderly people had the highest death rates, even though this was the only group that had any prior immunity, and there was a second wave in this group in 1958.

The new H2N2 virus completely replaced the previous H1N1 type, and became the new seasonal influenza type.

The “Hong Kong Flu” of 1968 – 1969

This pandemic started in mid-1968 in Hong Kong, and rapidly spread in a few months to India, the Philippines, Australia, Europe and the USA. By 1969, it had reached Japan, Africa and South America. Worldwide, the death toll peaked in December – January. However, although around one million people died, the death rate was lower than in 1957 – 1958 for a number of reasons, including the following:

The virus was similar in some respects to the Asian Flu variant – it was an H3N2 isolate, similar to the pre-1918 seasonal type, sharing N2 – meaning people infected then had partial immunity

The better availability of antibiotics meant secondary bacterial infections were less of a problem.

A vaccine to the new virus became available a month after the epidemic peaked in the USA – following a trend which had started with the 1958 pandemic, of vaccines becoming available only after the peak of the pandemic had passed.

An interesting development soon after this was the finding that waterfowl are the natural hosts of all influenza A viruses – and that there was a greater diversity of viruses in birds than in humans.

The “Red Flu” of 1977

Between May and November of 1977, an epidemic of influenza spread out of north-eastern China and the former Soviet Union – hence the name “Red Flu”. The disease was, however, limited to people under the age of 25 – and was generally mild. It was soon found that virus responsible was effectively identical to the H1N1 that had circulated from 1918 through to 1958, and which had been replaced by the Asian flu, which was in turn supplanted by the Hong Kong flu. This was a most unlikely scenario, given that it was already known that influenza A viruses mutated rapidly as they multiplied – and it had been twenty years since the Spanish or H1N1 flu had been seen in humans. It also explained why infections were limited to young people: anyone who had caught the seasonal flu prior to 1958 was protected.

There has been speculation that the pandemic was due to an inadequately-inactivated or attenuated vaccine released in a trial; there has even been mention of escape from a freezer in a biological warfare lab. There is no firm evidence for either possibility; however, the result is that the virus that had reappeared then co-circulated with the H3N2 as a seasonal virus, continuously until the next pandemic. This was unusual, as a pandemic virus usually becomes the next seasonal strain.

The “Swine Flu” of 2009

The next major pandemic to follow on from the 1968 outbreak was again a type A H1N1 virus – which this time, originated in Mexico or the south-western USA, and probably came directly from intensively-farmed pigs. This had been an unusually long interval between pandemics, and warnings of the coming plague had been issued regularly for years: however, it had been expected that the next pandemic would involve the highly pathogenic avian influenza virus H5N1, which had been popping up since 1997, and had been established as an endemic virus in farmed chickens since 2004. This was therefore rather a surprise – but a reasonably welcome one, as the virus turned out to be relatively mild in its effects.

Intensive research on the origin of the virus threw up some very interesting results: it was effectively a direct descendant of the original Spanish flu H1N1 virus, but which had been circulating in pigs ever since 1918 – and had had contributions of genetic material from swine, humans and birds (see Chapter 3, here).

By June 2009 the World Health Organisation had raised the pandemic alert level to Phase 6 – the highest level, indicating that the virus had spread worldwide and that there were infected people in most countries. The “swine flu” pandemic was not as serious as had been feared, however: symptoms of infection were similar to seasonal influenza, albeit with a greater incidence of diarrhoea and vomiting. The virus was also found to preferentially bind to cells deeper in the lungs than seasonal viruses: this explained both why it was generally mild – it did not often get that far down – but also why it could be fatal, as it could cause severe and sudden pneumonia if it did penetrate deep enough, similar to the 1918 influenza. Binding to cells in the intestines also explained the unusual nausea and vomiting. It was also found that there were distinct high-risk groups, including pregnant women and obese individuals. In these respects it was similar to the 1918 flu, as this also predominantly affected young people, and pregnant mothers.

Vaccine manufacture was initiated in June 2009 by the WHO and manufacturers: while there was some concern over the slower-than-normal growth rate of the vaccine strains of the virus, this was rectified in a few months. However, as also happened with the other pandemics, there was not enough vaccine made soon enough to deal effectively with the pandemic – even though similarities between the pandemic virus and the 1977 outbreak virus meant that most middle-aged people had pre-existing immunity to it, which either prevented infection, or reduced the severity of infections. This also meant a single dose was sufficient in adults, similar to the seasonal vaccine.

While the disease may have been mild in most cases, and initially the death toll was thought to be low, by 2012 it was calculated that 300 000 or more people probably died, mainly in Africa and Southeast Asia. A sobering quote: “since the people who died were much younger than is normally the case from influenza, in terms of years of life lost the H1N1 pandemic was significantly more lethal than the raw numbers suggest”. The virus has now become a normal seasonal strain, replacing the previously-circulating H1N1, but interestingly, has not replaced the H3N2 that has circulated since 1968.

All material Copyright EP Rybicki, except for the Camp Funston image, which is in the public domain.

“A key observation about the human immune response to repeated exposure to influenza A is that the first strain infecting an individual apparently produces the strongest adaptive immune response. Although antibody titers measure that response, the interpretation of titers to multiple strains – from the same sera – in terms of infection history is clouded by age effects, cross reactivity and immune waning. From July to September 2009, we collected serum samples from 151 residents of Guangdong Province, China, 7 to 81 years of age. Neutralization tests were performed against strains representing six antigenic clusters of H3N2 influenza circulating between 1968 and 2008, and three recent locally circulating strains. Patterns of neutralization titers were compared based on age at time of testing and age at time of the first isolation of each virus. Neutralization titers were highest for H3N2 strains that circulated in an individual’s first decade of life (peaking at 7 years). Further, across strains and ages at testing, statistical models strongly supported a pattern of titers declining smoothly with age at the time a strain was first isolated. Those born 10 or more years after a strain emerged generally had undetectable neutralization titers to that strain (<1:10). Among those over 60 at time of testing, titers tended to increase with age. The observed pattern in H3N2 neutralization titers can be characterized as one of antigenic seniority: repeated exposure and the immune response combine to produce antibody titers that are higher to more ‘senior’ strains encountered earlier in life.”

An interesting paper, which helps explain several observations made over the years with pandemic flu: for example, in the 2009 H1N1 pandemic, older people seemed to be more protected – and rhe same was probably true of the 1918 pandemic.

“IntroductionThe publication in this issue of these research papers on the airborne tranimssion [sic] of H5N1 marks the end of 8 months of controversy over whether some of the data, now freely accessible, should be withheld in the public interest.”

I think this is an important landmark in the so-called “dual use” debate: that is, the propensity of bodies in the US to attempt to regulate the release of information that MAY be usable in the making of bioweapons, or be usable in bioterror attacks.

Let us diffidently point out at this juncture that it is only really the superpowers who are definitively known in recent years to have had bioweapons programmes – apart from apartheid-era South Africa, that is! – and that damn nearly ANYTHING published on transmission or mechanisms of pathogenicity of human or animal pathogens (or even plant, for that matter) could be termed “dual use” if someone wanted to – and censored as a result.

It is also – as I tire of pointing out – possible to PROTECT against H5NX viruses using conventional vaccines right now – and the new universal flu vaccines coming on stream will almost certainly make this even more feasible.

The fact is that H5N1 flu is an ever-present threat to people living in Egypt, Indonesia, Cambodia, Viet Nam, Thailand and China – WITHOUT being weaponised. It is no more than a notional threat to the US or Europe – and keeping information that could help in understanding how or how soon the virus could mutate to pandemicity out of people’s hands, is simply stupid.

“A new Swedish study shows that all Swedes who developed narcolepsy from the swine flu vaccine Pandemrix received the vaccine from 12 of the 35 batches, despite the claim by the responsible agency that no such connection exists.”

There are some slightly disturbing connections between the H1N1 2009 pdm virus and narcolepsy: the virus itself seems to have caused narcolepsy in some of those infected; now a vaccine is implicated – is this an innate property of certain of the virus proteins, possibly?

An international research team has manufactured a new protein that can combat deadly flu epidemics.

The paper, featured on the cover of the current issue of Nature Biotechnology, demonstrates ways to use manufactured genes as antivirals, which disable key functions of the flu virus, said Tim Whitehead, assistant professor of chemical engineering and materials science at Michigan State University.